Process chromatography in its many variant forms has become the dominant downstream processing tool for difficult separations in biotechnology, but it is inherently expensive and is not used to a significant extent for commercial scale separations in any other industry. In particular, process chromatography is not used significantly in food processing or petrochemical technology. Chromatography depends upon concentration diffusion between stationary and mobile phases, and, as commercial interest shifts toward larger substrates such as plasmids and viruses, diffusion tends to become slower and to make separations increasingly difficult.
At the same time, many other potentially competitive techniques have been developing, and engineers have finally begun to show real initiative for process development in a variety of biological applications. Lightfoot, E. N., and J. S. Moscariello, 2004, Bioseparations, Biotech. and Bioeng. 87: 259-273. Increasingly efficient renaturation of proteins from inclusion bodies shows promise of replacing the capture steps now performed by batch adsorption chromatography in a variety of applications, and crystallization appears to be increasing in importance for finer separations. Simulated moving beds are receiving increased attention.
Membrane filtrations are already providing increased competition to chromatography for the polishing stages of downstream processing, and they are becoming more and more selective, even for such large molecules as proteins. Cheang, B., and A. L. Zydney, 2004, A two-stage ultrafiltration process for fractionation of whey protein isolate, J. Mem. Sci. 231: 159-167. Several investigators report the use of simple two-stage cascades, but these cascades do not incorporate counterflow principles.
There is also increasing interest in continuous downstream processing for which chromatography is ill suited. Use of simulated moving beds, the only continuous process currently available, is both cumbersome and poorly suited to feedback control. To date, these devices have been limited to very clean stable systems, for example in the resolution of enantiomers from highly purified racemic mixtures. Finally, there is increasing interest in larger entities such as nucleic acids and viruses, and these have such low diffusivities that the choice of suitable adsorbents is severely limited. Pressure induced flow across selective membranes however can increase transport rates by convection relative to those for diffusion alone. Bird, R. B., W. E. Stewart, and E. N. Lightfoot. 2002, “Transport Phenomena”, Wiley.
A new look at downstream processing is warranted, and membrane cascades may provide new and important methods for separating components from mixtures. Membrane selectivities are rapidly increasing, and there is now a wealth of practical operating experience available for purposes of preliminary design. Moreover, membranes are available for dealing with an extremely wide range of molecular weights, from small monomeric molecules to mammalian cells. Moreover, the technology of dealing with membrane cascades was very highly developed during the 1940s in connection with the effusion process for uranium isotope fractionation. Von Halle, E, and J. Schachter, 1998, “Diffusion Separation Methods”, in Kirk-Othmer Encyclopedia of Chemical Technology, 4th Ed., J. Kroschwitz, Ed., Wiley. Even very simple cascades have not been widely used in biotechnology, however, in large part because of control problems and lack of operating experience. Membrane cascades thus present a promising field for research and development. A logical starting point for investigation will be the ideal cascade theory of isotope separations. As described below, isotope separations have much in common with potential biological applications.
It is desirable to start with simple prototype systems and then move by degrees to more complex systems in more promising situations. Fortunately, there are some simple applications where useful results can be obtained rather simply. One can then gain experience and at the same time produce economic processes. There are guides in the literature to aid in this stepwise approach. There are several examples of essentially binary protein solutions (e.g., Cheang and Zydney, J. Mem. Sci. 231 (2004)). Another logical starting point would be the tryptophan resolution of Romero and Zydney as the components are inexpensive and stable, and assays are unusually simple. Moreover, one needs only an ultrafiltration membrane under situations where sensitivity to minor changes in behavior is probably insignificant-one can concentrate here on solvent problems and development of a reliable control strategy. One can then proceed to other well-documented and simple separations such as removal of dimers from monomeric bovine serum albumin (BSA). After that, one can begin in earnest on systems where a more complex cascade is particularly desired.
Many cascade separation systems have been proposed, but none apparently incorporating the ideal cascade approach where design strategy is divided into: (i) separation of the solutes of interest using a solvent-free description; and (ii) solvent management.